Antibody formulation

ABSTRACT

The invention provides a stable aqueous pharmaceutical formulation comprising a therapeutically effective amount of an antibody, optionally, not subjected to prior lyophilization, a buffer maintaining the pH in the range from about 4.0 to about 6.0, and an optional surfactant, methods for making such a formulation, and methods of using such a formulation.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/288,535, filed Dec. 21, 2009, the disclosure of whichis hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention is directed to a stable aqueous pharmaceuticalformulation comprising an antibody.

BACKGROUND

In the past years, advances in biotechnology have made it possible toproduce a variety of proteins for pharmaceutical applications usingrecombinant DNA techniques. Because proteins are larger and more complexthan traditional organic and inorganic drugs (e.g., possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such proteins poses special problems. For a proteinto remain biologically active, a formulation must preserve intact theconformational integrity of at least a core sequence of the protein'samino acids while at the same time protecting the protein's multiplefunctional groups from degradation. Degradation pathways for proteinscan involve chemical instability (e.g., any process which involvesmodification of the protein by bond formation or cleavage resulting in anew chemical entity) or physical instability (e.g., changes in thehigher order structure of the protein). Chemical instability can resultfrom deamidation, racemization, hydrolysis, oxidation, beta eliminationor disulfide exchange. Physical instability can result fromdenaturation, aggregation, precipitation or adsorption, for example. Thethree most common protein degradation pathways are protein aggregation,deamidation and oxidation. Cleland et al Critical Reviews in TherapeuticDrug Carrier Systems 10(4): 307-377 (1993).

Included in the proteins used for pharmaceutical applications areantibodies. An example of an antibody useful for therapy is an antibodywhich binds to anti-VEGF. There is a need in the art for a stableaqueous pharmaceutical formulation comprising an antibody, such as ananti-VEGF antibody, which is suitable for therapeutic use.

SUMMARY

The invention provides stable aqueous pharmaceutical formulationscomprising

a therapeutically effective amount of an antibody, optionally, notsubjected to prior lyophilization, a buffer maintaining the pH in therange from about 4.0 to about 6.0, and an optional surfactant, methodsof making the formulation and methods of using the formulation.

One embodiment of the invention provides a stable aqueous pharmaceuticalformulation, the formulation comprising a therapeutically effectiveamount of an antibody in an arginine buffer, pH 4.0 to 6.0. In someembodiments, the buffer is an arginine acetate buffer, pH 4.5 to 5.5. Insome embodiments, the buffer is an arginine acetate buffer, pH 4.8 to5.4. In some embodiments, the buffer is an arginine acetate buffer, pH5.2. In some embodiments, the arginine actetate concentration in thebuffer is from about 25 mM to about 250 mM. In some embodiments, thearginine actetate concentration in the buffer is from about 50 mM toabout 250 mM. In some embodiments, the arginine actetate concentrationin the buffer is from about 75 mM to about 250 mM. In some embodiments,the arginine actetate concentration in the buffer is from about 100 mMto about 250 mM. In some embodiments, the arginine acetate concentrationin the buffer is from about 120 mM to about 240 mM. In some embodiments,the arginine acetate concentration in the buffer is from about 150 mM toabout 225 mM. In some embodiments, the arginine acetate concentration inthe buffer is about 200 mM. In some embodiments, the formulation furthercomprises a surfactant. In some embodiments, the surfactant ispolysorbate. In some embodiments, the polysorbate is polysorbate 20. Insome embodiments, the surfactant concentration is from 0.0001% to about1.0%. In some embodiments, the surfactant concentration is from about0.01% to about 0.05%. In some embodiments, the surfactant concentrationis 0.04%. In some embodiments, the antibody concentration is from about10 mg/ml to about 250 mg/ml. In some embodiments, the antibodyconcentration is from about 25 mg/ml to 200 mg/ml. In some embodiments,the antibody concentration is from about 30 mg/ml to 175 mg/ml. In someembodiments, the antibody concentration is from about 50 mg/ml to about150 mg/ml. In some embodiments, the antibody concentration is from about75 mg/ml to about 125 mg/ml. In some embodiments, the antibodyconcentration is from about 25 mg/ml to about 100 mg/ml. In someembodiments, the antibody is not subject to prior lyophilization. Insome embodiments, the antibody binds VEGF. In some embodiments, theantibody is a monoclonal antibody. In some embodiments, the monoclonalantibody is a full length antibody. In some embodiments, the monoclonalantibody is an IgG1 antibody. In some embodiments, the monoclonalantibody is a humanized antibody. In some embodiments, the monoclonalantibody is an antibody fragment comprising an antigen-binding region.In some embodiments, the antibody fragment is a Fab or F(ab′)2 fragment.In some embodiments, the monoclonal antibody binds VEGF. In someembodiments, the antibody is bevacizumab. In some embodiments, themonoclonal antibody is susceptible to aggregation. In some embodiments,the buffer is 200 mM arginine acetate pH 5.2, the surfactant ispolysorbate in an amount of about 0.01-0.1% v/v and the formulation isstable at a temperature of about 40° C. for at least 28 days. In someembodiments, the formulation is sterile. In some embodiments, theformulation is stable upon storage at about 40° C. for at least 28 days.In some embodiments, the formulation is aqueous and is administered to asubject. In some embodiments, the formulation is for intravenous (IV),subcutaneous (SQ) or intramuscular (IM) administration. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml.

Another embodiment of the invention provides an article of manufacturecomprising a container holding a stable aqueous pharmaceuticalformulation comprising a therapeutically effective amount of anantibody, an arginine acetate buffer from about pH 4.5 to about 6.0, anda surfactant. In some embodiments, the antibody concentration is fromabout 10 mg/ml to about 250 mg/ml. In some embodiments, the antibodyconcentration is from about 25 mg/ml to 200 mg/ml. In some embodiments,the antibody concentration is from about 30 mg/ml to 175 mg/ml. In someembodiments, the antibody concentration is from about 50 mg/ml to about150 mg/ml. In some embodiments, the antibody concentration is from about75 mg/ml to about 125 mg/ml. In some embodiments, the antibodyconcentration is from about 25 mg/ml to about 100 mg/ml. In someembodiments, the antibody is not subject to prior lyophilization. Insome embodiments, the antibody binds VEGF. In some embodiments, theantibody is a monoclonal antibody. In some embodiments, the monoclonalantibody is a full length antibody. In some embodiments, the monoclonalantibody is an IgG1 antibody. In some embodiments, the monoclonalantibody is a humanized antibody. In some embodiments, the monoclonalantibody is an antibody fragment comprising an antigen-binding region.In some embodiments, the antibody fragment is a Fab or F(ab′)2 fragment.In some embodiments, the monoclonal antibody binds VEGF. In someembodiments, the antibody is bevacizumab. In some embodiments, themonoclonal antibody is susceptible to aggregation. In some embodiments,the arginine actetate concentration in the buffer is from about 25 mM toabout 250 mM. In some embodiments, the arginine actetate concentrationin the buffer is from about 50 mM to about 250 mM. In some embodiments,the arginine actetate concentration in the buffer is from about 75 mM toabout 250 mM. In some embodiments, the arginine actetate concentrationin the buffer is from about 100 mM to about 250 mM. In some embodiments,the arginine acetate concentration in the buffer is from about 120 mM toabout 240 mM. In some embodiments, the arginine acetate concentration inthe buffer is from about 150 mM to about 225 mM. In some embodiments,the arginine acetate concentration in the buffer is about 200 mM. Insome embodiments, the arginine acetate buffer has a pH from about 4.5 toabout 5.5. In some embodiments, the arginine acetate buffer has a pHfrom about 4.8 to about 5.4. In some embodiments, the arginine acetatebuffer has a pH of about 5.2. In some embodiments, the surfactant ispolysorbate. In some embodiments, the polysorbate is polysorbate 20. Insome embodiments, the surfactant concentration is from 0.0001% to about1.0%. In some embodiments, the surfactant concentration is from about0.01% to about 0.05%. In some embodiments, the surfactant concentrationis 0.04%. In some embodiments, the formulation is sterile. In someembodiments, the formulation is stable upon storage at about 40° C. forat least 28 days. In some embodiments, the formulation is aqueous and isadministered to a subject. In some embodiments, the formulation is forintravenous (IV), subcutaneous (SQ) or intramuscular (IM)administration. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 25 mg/ml toabout 175 mg/ml. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 50 mg/ml toabout 150 mg/ml. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 75 mg/ml toabout 125 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 25 mg/ml toabout 175 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 50 mg/ml toabout 150 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 75 mg/ml toabout 125 mg/ml. In some embodiments, the formulation is for IMadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the formulation is for IMadministration and the antibody concentration is from about 25 mg/ml toabout 175 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 50 mg/ml toabout 150 mg/ml. In some embodiments, the formulation is for IMadministration and the antibody concentration is from about 75 mg/ml toabout 125 mg/ml.

A further embodiment of the invention provides a method for stabilizingan antibody in an aqueous pharmaceutical formulation by combining atherapeutically effective amount of an antibody, an arginine acetatebuffer from about pH 4.5 to about 6.0, and a surfactant. In someembodiments, the antibody concentration is from about 10 mg/ml to about250 mg/ml. In some embodiments, the antibody concentration is from about25 mg/ml to 200 mg/ml. In some embodiments, the antibody concentrationis from about 30 mg/ml to 175 mg/ml. In some embodiments, the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the antibody concentration is from about 75 mg/ml to about125 mg/ml. In some embodiments, the antibody concentration is from about25 mg/ml to about 100 mg/ml. In some embodiments, the antibody is notsubject to prior lyophilization. In some embodiments, the antibody bindsVEGF. In some embodiments, the antibody is a monoclonal antibody. Insome embodiments, the monoclonal antibody is a full length antibody. Insome embodiments, the monoclonal antibody is an IgG1 antibody. In someembodiments, the monoclonal antibody is a humanized antibody. In someembodiments, the monoclonal antibody is an antibody fragment comprisingan antigen-binding region. In some embodiments, the antibody fragment isa Fab or F(ab′)2 fragment. In some embodiments, the monoclonal antibodybinds VEGF. In some embodiments, the antibody is bevacizumab. In someembodiments, the monoclonal antibody is susceptible to aggregation. Insome embodiments, the arginine actetate concentration in the buffer isfrom about 25 mM to about 250 mM. In some embodiments, the arginineactetate concentration in the buffer is from about 50 mM to about 250mM. In some embodiments, the arginine actetate concentration in thebuffer is from about 75 mM to about 250 mM. In some embodiments, thearginine actetate concentration in the buffer is from about 100 mM toabout 250 mM. In some embodiments, the arginine acetate concentration inthe buffer is from about 120 mM to about 240 mM. In some embodiments,the arginine acetate concentration in the buffer is from about 150 mM toabout 225 mM. In some embodiments, the arginine acetate concentration inthe buffer is about 200 mM. In some embodiments, the arginine acetatebuffer has a pH from about 4.5 to about 5.5. In some embodiments, thearginine acetate buffer has a pH from about 4.8 to about 5.4. In someembodiments, the arginine acetate buffer has a pH of about 5.2. In someembodiments, the surfactant is polysorbate. In some embodiments, thepolysorbate is polysorbate 20. In some embodiments, the surfactantconcentration is from 0.0001% to about 1.0%. In some embodiments, thesurfactant concentration is from about 0.01% to about 0.05%. In someembodiments, the surfactant concentration is 0.04%. In some embodiments,the formulation is sterile. In some embodiments, the formulation isstable upon storage at about 40° C. for at least 28 days. In someembodiments, the formulation is aqueous and is administered to asubject. In some embodiments, the formulation is for intravenous (IV),subcutaneous (SQ) or intramuscular (IM) administration. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml.

Yet another embodiment of the invention provides a stable aqueouspharmaceutical formulation comprising a therapeutically effective amountof an antibody, 200 mM arginine acetate buffer at pH 5.2, and asurfactant. In some embodiments, the antibody concentration is fromabout 10 mg/ml to about 250 mg/ml. In some embodiments, the antibodyconcentration is from about 25 mg/ml to 200 mg/ml. In some embodiments,the antibody concentration is from about 30 mg/ml to 175 mg/ml. In someembodiments, the antibody concentration is from about 50 mg/ml to about150 mg/ml. In some embodiments, the antibody concentration is from about75 mg/ml to about 125 mg/ml. In some embodiments, the antibodyconcentration is from about 25 mg/ml to about 100 mg/ml. In someembodiments, the antibody is not subject to prior lyophilization. Insome embodiments, the antibody binds VEGF. In some embodiments, theantibody is a monoclonal antibody. In some embodiments, the monoclonalantibody is a full length antibody. In some embodiments, the monoclonalantibody is an IgG1 antibody. In some embodiments, the monoclonalantibody is a humanized antibody. In some embodiments, the monoclonalantibody is an antibody fragment comprising an antigen-binding region.In some embodiments, the antibody fragment is a Fab or F(ab′)2 fragment.In some embodiments, the monoclonal antibody binds VEGF. In someembodiments, the antibody is bevacizumab. In some embodiments, themonoclonal antibody is susceptible to aggregation. In some embodiments,the surfactant is polysorbate. In some embodiments, the polysorbate ispolysorbate 20. In some embodiments, the surfactant concentration isfrom 0.0001% to about 1.0%. In some embodiments, the surfactantconcentration is from about 0.01% to about 0.05%. In some embodiments,the surfactant concentration is 0.04%. In some embodiments, theformulation is sterile. In some embodiments, the formulation is stableupon storage at about 40° C. for at least 28 days. In some embodiments,the formulation is aqueous and is administered to a subject. In someembodiments, the formulation is for intravenous (IV), subcutaneous (SQ)or intramuscular (IM) administration. In some embodiments, theformulation is for IV administration and the antibody concentration isfrom about 10 mg/ml to about 250 mg/ml. In some embodiments, theformulation is for IV administration and the antibody concentration isfrom about 25 mg/ml to about 175 mg/ml. In some embodiments, theformulation is for IV administration and the antibody concentration isfrom about 50 mg/ml to about 150 mg/ml. In some embodiments, theformulation is for IV administration and the antibody concentration isfrom about 75 mg/ml to about 125 mg/ml. In some embodiments, theformulation is for SQ administration and the antibody concentration isfrom about 10 mg/ml to about 250 mg/ml. In some embodiments, theformulation is for SQ administration and the antibody concentration isfrom about 25 mg/ml to about 175 mg/ml. In some embodiments, theformulation is for SQ administration and the antibody concentration isfrom about 50 mg/ml to about 150 mg/ml. In some embodiments, theformulation is for SQ administration and the antibody concentration isfrom about 75 mg/ml to about 125 mg/ml. In some embodiments, theformulation is for IM administration and the antibody concentration isfrom about 10 mg/ml to about 250 mg/ml. In some embodiments, theformulation is for IM administration and the antibody concentration isfrom about 25 mg/ml to about 175 mg/ml. In some embodiments, theformulation is for SQ administration and the antibody concentration isfrom about 50 mg/ml to about 150 mg/ml. In some embodiments, theformulation is for IM administration and the antibody concentration isfrom about 75 mg/ml to about 125 mg/ml.

A further embodiment of the invention provides a pharmaceuticalformulation comprising: (a) a full length IgG1 antibody susceptible todeamidation or aggregation in an amount from about 10 mg/mL to about 250mg/mL; (b) arginine acetate buffer, pH 4.5 to 6.0; and (c) polysorbate20 in an amount from about 0.01% to about 0.1%. In some embodiments, theantibody concentration is from about 25 mg/ml to 200 mg/ml. In someembodiments, the antibody concentration is from about 30 mg/ml to 175mg/ml. In some embodiments, the antibody concentration is from about 50mg/ml to about 150 mg/ml. In some embodiments, the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the antibody concentration is from about 25 mg/ml to about100 mg/ml. In some embodiments, the antibody is not subject to priorlyophilization. In some embodiments, the antibody binds VEGF. In someembodiments, the antibody is bevacizumab. In some embodiments, theantibody is a humanized antibody. In some embodiments, the arginineactetate concentration in the buffer is from about 25 mM to about 250mM. In some embodiments, the arginine actetate concentration in thebuffer is from about 50 mM to about 250 mM. In some embodiments, thearginine actetate concentration in the buffer is from about 75 mM toabout 250 mM. In some embodiments, the arginine actetate concentrationin the buffer is from about 100 mM to about 250 mM. In some embodiments,the arginine acetate concentration in the buffer is from about 120 mM toabout 240 mM. In some embodiments, the arginine acetate concentration inthe buffer is from about 150 mM to about 225 mM. In some embodiments,the arginine acetate concentration in the buffer is about 200 mM. Insome embodiments, the arginine acetate buffer has a pH from about 4.5 toabout 5.5. In some embodiments, the arginine acetate buffer has a pHfrom about 4.8 to about 5.4. In some embodiments, the arginine acetatebuffer has a pH of about 5.2. In some embodiments, the polysorbate 20 isfrom about 0.01% to about 0.05%. In some embodiments, the polysorbate 20is 0.04%. In some embodiments, the formulation is sterile. In someembodiments, the formulation is stable upon storage at about 40° C. forat least 28 days. In some embodiments, the formulation is aqueous and isadministered to a subject. In some embodiments, the formulation is forintravenous (IV), subcutaneous (SQ) or intramuscular (IM)administration. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 25 mg/ml toabout 175 mg/ml. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 50 mg/ml toabout 150 mg/ml. In some embodiments, the formulation is for IVadministration and the antibody concentration is from about 75 mg/ml toabout 125 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 25 mg/ml toabout 175 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 50 mg/ml toabout 150 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 75 mg/ml toabout 125 mg/ml. In some embodiments, the formulation is for IMadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the formulation is for IMadministration and the antibody concentration is from about 25 mg/ml toabout 175 mg/ml. In some embodiments, the formulation is for SQadministration and the antibody concentration is from about 50 mg/ml toabout 150 mg/ml. In some embodiments, the formulation is for IMadministration and the antibody concentration is from about 75 mg/ml toabout 125 mg/ml.

Yet another embodiment of the invention provides a pharmaceuticalformulation comprising an antibody that binds to VEGF in an arginineacetate buffer at a pH from about 4.5 to about 6.0, and a surfactant. Insome embodiments, the antibody concentration is from about 10 mg/ml toabout 250 mg/ml. In some embodiments, the antibody concentration is fromabout 25 mg/ml to 200 mg/ml. In some embodiments, the antibodyconcentration is from about 30 mg/ml to 175 mg/ml. In some embodiments,the antibody concentration is from about 50 mg/ml to about 150 mg/ml. Insome embodiments, the antibody concentration is from about 75 mg/ml toabout 125 mg/ml. In some embodiments, the antibody concentration is fromabout 25 mg/ml to about 100 mg/ml. In some embodiments, the antibody isnot subject to prior lyophilization. In some embodiments, the antibodyis a monoclonal antibody. In some embodiments, the monoclonal antibodyis a full length antibody. In some embodiments, the monoclonal antibodyis an IgG1 antibody. In some embodiments, the monoclonal antibody is ahumanized antibody. In some embodiments, the monoclonal antibody is anantibody fragment comprising an antigen-binding region. In someembodiments, the antibody fragment is a Fab or F(ab′)2 fragment. In someembodiments, the antibody is bevacizumab. In some embodiments, themonoclonal antibody is susceptible to aggregation. In some embodiments,the arginine actetate concentration in the buffer is from about 25 mM toabout 250 mM. In some embodiments, the arginine actetate concentrationin the buffer is from about 50 mM to about 250 mM. In some embodiments,the arginine actetate concentration in the buffer is from about 75 mM toabout 250 mM. In some embodiments, the arginine acetate concentration inthe buffer is from about 120 mM to about 240 mM. In some embodiments,the arginine acetate concentration in the buffer is from about 150 mM toabout 225 mM. In some embodiments, the arginine acetate concentration inthe buffer is about 200 mM. In some embodiments, the arginine acetatebuffer has a pH from about 4.5 to about 5.5. In some embodiments, thearginine acetate buffer has a pH from about 4.8 to about 5.4. In someembodiments, the arginine acetate buffer has a pH of about 5.2. In someembodiments, the surfactant is polysorbate. In some embodiments, thepolysorbate is polysorbate 20. In some embodiments, the surfactantconcentration is from 0.0001% to about 1.0%. In some embodiments, thesurfactant concentration is from about 0.01% to about 0.05%. In someembodiments, the surfactant concentration is 0.04%. In some embodiments,the formulation is sterile. In some embodiments, the formulation isstable upon storage at about 40° C. for at least 28 days. In someembodiments, the formulation is aqueous and is administered to asubject. In some embodiments, the formulation is for intravenous (IV),subcutaneous (SQ) or intramuscular (IM) administration. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml.

Another embodiment of the invention provides a method for reducingaggregation of a therapeutic monoclonal antibody, comprising formulatingthe antibody in an arginine acetate buffer, pH 4.5 to 6.0. In someembodiments, the antibody concentration is from about 10 mg/ml to about250 mg/ml. In some embodiments, the antibody concentration is from about25 mg/ml to 200 mg/ml. In some embodiments, the antibody concentrationis from about 30 mg/ml to 175 mg/ml. In some embodiments, the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the antibody concentration is from about 75 mg/ml to about125 mg/ml. In some embodiments, the antibody concentration is from about25 mg/ml to about 100 mg/ml. In some embodiments, the antibody is notsubject to prior lyophilization. In some embodiments, the antibody is amonoclonal antibody. In some embodiments, the monoclonal antibody is afull length antibody. In some embodiments, the monoclonal antibody is anIgG1 antibody. In some embodiments, the monoclonal antibody is ahumanized antibody. In some embodiments, the monoclonal antibody is anantibody fragment comprising an antigen-binding region. In someembodiments, the antibody fragment is a Fab or F(ab′)2 fragment. In someembodiments, the monoclonal antibody binds VEGF. In some embodiments,the antibody is bevacizumab. In some embodiments, the monoclonalantibody is susceptible to aggregation. In some embodiments, thearginine actetate concentration in the buffer is from about 25 mM toabout 250 mM. In some embodiments, the arginine actetate concentrationin the buffer is from about 50 mM to about 250 mM. In some embodiments,the arginine actetate concentration in the buffer is from about 75 mM toabout 250 mM. In some embodiments, the arginine acetate concentration inthe buffer is from about 120 mM to about 240 mM. In some embodiments,the arginine acetate concentration in the buffer is from about 150 mM toabout 225 mM. In some embodiments, the arginine acetate concentration inthe buffer is about 200 mM. In some embodiments, the arginine acetatebuffer has a pH from about 4.5 to about 5.5. In some embodiments, thearginine acetate buffer has a pH from about 4.8 to about 5.4. In someembodiments, the arginine acetate buffer has a pH of about 5.2. In someembodiments, the formulation is sterile. In some embodiments, theformulation is stable upon storage at about 40° C. for at least 28 days.In some embodiments, the formulation is aqueous and is administered to asubject. In some embodiments, the formulation is for intravenous (IV),subcutaneous (SQ) or intramuscular (IM) administration. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IV administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 10 mg/ml to about 250 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 25 mg/ml to about 175 mg/ml. In someembodiments, the formulation is for SQ administration and the antibodyconcentration is from about 50 mg/ml to about 150 mg/ml. In someembodiments, the formulation is for IM administration and the antibodyconcentration is from about 75 mg/ml to about 125 mg/ml.

Even a further embodiment of the invention provides an article ofmanufacture comprising a container holding any one of the formulationsdescribed herein.

Yet a further embodiment of the invention provides a vial with a stopperpierceable by a syringe comprising any one of the formulations describedherein. In some embodiments, the vial is stored at about 2-8° C. In someembodiments, the vial is a 3 cc, 20 cc or 50 cc vial.

Another embodiment of the invention provides a stainless steel tankcomprising any one of the formulations described herein inside the tank.In some embodiments, the formulation is frozen.

A further embodiment of the invention provides a method of making apharmaceutical formulation comprising: (a) preparing any one of theformulations described herein; and (b) evaluating physical stability,chemical stability, or biological activity of the antibody in theformulation.

Yet another embodiment of the invention provides a method of treating adisease or disorder in a subject comprising administering any one of theformulations described herein to a subject in an amount effective totreat the disease or disorder. In some embodiments, the disease iscancer. In some embodiments, the cancer is selected from colorectalcancer, lung cancer, breast cancer, renal cancer, and glioblastoma.

These and other embodiments of the invention are further described bythe detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates total aggregate levels detected in anti-VEGFformulations (100 mg/ml) stored for up to 4 weeks at 40° C.

FIG. 2 illustrates total aggregate levels detected in anti-VEGFformulations (0, 50, 100, and 150 mg/ml).

FIG. 3 illustrates the dimer levels detected in anti-VEGF formulations(100 mg/ml) compared to stored for up to 4 weeks at 40° C.

FIG. 4 illustrates the viscosity of anti-VEGF formulations at 20° C. asa function of anti-VEGF concentration (25, 50, 100, 125, 150, or 175mg/ml).

DETAILED DESCRIPTION I. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, the singular forms “a”, “an” and“the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to “a molecule” optionallyincludes a combination of two or more such molecules, and the like.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile. “Pharmaceuticallyacceptable” excipients (vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

A “sterile” formulation is asceptic or free or essentially free from allliving microorganisms and their spores.

Herein, a “frozen” formulation is one at a temperature below 0° C.Generally, the frozen formulation is not freeze-dried, nor is itsubjected to prior, or subsequent, lyophilization. In certainembodiments, the frozen formulation comprises frozen drug substance forstorage (in stainless steel tank) or frozen drug product (in final vialconfiguration).

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Preferably, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage. The storage period is generallyselected based on the intended shelf-life of the formulation. Variousanalytical techniques for measuring protein stability are available inthe art and are reviewed in Peptide and Protein Drug Delivery, 247-301,Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) andJones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.Stability can be measured at a selected temperature for a selected timeperiod. In certain embodiments, the formulation is stable at about 40°C. for at least about 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, or more days. Incertain embodiments, the formulation is stable at about 40° C. for atleast about 1, 2, 3, 4, 5, 6, 7, 8, or more weeks. In certainembodiments, the formulation is stable at about 25° C. for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or more months. In certain embodiments, the formulation isstable at about 5° C. for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or more months. Incertain embodiments, the formulation is stable at about −20° C. for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. In certainembodiments, the formulation is stable at 5° C. or −20° C. for at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more months. Furthermore, theformulation is preferably stable following freezing (to, e.g., −20° C.or −70° C.) and thawing of the formulation, for example following 1, 23, 4, or 5 cycles of freezing and thawing. Stability can be evaluatedqualitatively and/or quantitatively in a variety of different ways,including evaluation of aggregate formation (for example using sizeexclusion chromatography, by measuring turbidity, and/or by visualinspection); by assessing charge heterogeneity using cation exchangechromatography, image capillary isoelectric focusing (icIEF) orcapillary zone electrophoresis; amino-terminal or carboxy-terminalsequence analysis; mass spectrometric analysis; SDS-PAGE analysis tocompare reduced and intact antibody; peptide map (for example tryptic orLYS-C) analysis; evaluating biological activity or antigen bindingfunction of the antibody; etc. Instability may involve any one or moreof: aggregation, deamidation (e.g. Asn deamidation), oxidation (e.g. Metoxidation), isomerization (e.g. Asp isomeriation),clipping/hydrolysis/fragmentation (e.g. hinge region fragmentation),succinimide formation, unpaired cysteine(s), N-terminal extension,C-terminal processing, glycosylation differences, etc.

A protein “retains its physical stability” in a pharmaceuticalformulation if it shows no signs or very little of aggregation,precipitation and/or denaturation upon visual examination of colorand/or clarity, or as measured by UV light scattering or by sizeexclusion chromatography.

A protein “retains its chemical stability” in a pharmaceuticalformulation, if the chemical stability at a given time is such that theprotein is considered to still retain its biological activity as definedbelow. Chemical stability can be assessed by detecting and quantifyingchemically altered forms of the protein. Chemical alteration may involvesize modification (e.g. clipping) which can be evaluated using sizeexclusion chromatography, SDS-PAGE and/or matrix-assisted laserdesorption ionization/time-of-flight mass spectrometry (MALDI/TOF MS),for example. Other types of chemical alteration include chargealteration (e.g. occurring as a result of deamidation) which can beevaluated by ion-exchange chromatography or icIEF, for example.

An antibody “retains its biological activity” in a pharmaceuticalformulation, if the biological activity of the antibody at a given timeis within about 10% (within the errors of the assay) of the biologicalactivity exhibited at the time the pharmaceutical formulation wasprepared as determined in an antigen binding assay, for example. Other“biological activity” assays for antibodies are elaborated herein below.

Herein, “biological activity” of a monoclonal antibody refers to theability of the antibody to bind to antigen. It can further includeantibody binding to antigen and resulting in a measurable biologicalresponse which can be measured in vitro or in vivo. Such activity may beantagonistic or agonistic.

A “deamidated” monoclonal antibody herein is one in which one or moreasparagine residue thereof has been derivitized, e.g. to an asparticacid or an iso-aspartic acid.

An antibody which is “susceptible to deamidation” is one comprising oneor more residue, which has been found to be prone to deamidate.

An antibody which is “susceptible to aggregation” is one which has beenfound to aggregate with other antibody molecule(s), especially uponfreezing and/or agitation.

An antibody which is “susceptible to fragmentation” is one which hasbeen found to be cleaved into two or more fragments, for example at ahinge region thereof.

By “reducing deamidation, aggregation, or fragmentation” is intendedpreventing or decreasing the amount of deamidation, aggregation, orfragmentation relative to the monoclonal antibody formulated at adifferent pH or in a different buffer.

The antibody which is formulated is preferably essentially pure anddesirably essentially homogeneous (e.g., free from contaminatingproteins etc). “Essentially pure” antibody means a compositioncomprising at least about 90% by weight of the antibody, based on totalweight of the composition, preferably at least about 95% by weight.“Essentially homogeneous” antibody means a composition comprising atleast about 99% by weight of antibody, based on total weight of thecomposition.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Thebuffer of this invention preferably has a pH in the range from about 4.5to about 7.0, preferably from about 4.5 to about 6.5, for example from4.5 to 6.0, 4.5 to 5.9. 4.5 to 5.8, 4.5 to 5.7, 4.5 to 5.6, 4.5 to 5.5,4.5 to 5.6, 4.5 to 5.5, 4.5 to 5.4, 4.5 to 5.3, 4.5 to 5.2, 4.5 to 5.1,4.5 to 5.0, 4.5 to 4.9, 4.5 to 4.8, 4.5 to 4.7, or 4.5 to 4.6. In oneembodiment the buffer has a pH 4.5, 4.6, 4.7, 4.8, 4.8, 5.0, 5.1, 5.2,5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. Examples of buffers that willcontrol the pH in this range include acetate, succinate, succinate,gluconate, histidine, citrate, glycylglycine and other organic acidbuffers.

An “arginine buffer” is a buffer comprising arginine ions. Examples ofarginine buffers include arginine acetate, arginine chloride, argininephosphate, arginine sulfate, arginine succinate, etc. In one embodiment,the arginine buffer is arginine acetate. In the one embodiment, thearginine acetate buffer is prepared by titrating L-arginine (free base,solid) with acetic acid (liquid). In certain embodiments, the argininebuffer is at pH 4.5 to 6.0, 4.5 to 5.9. 4.5 to 5.8, 4.5 to 5.7, 4.5 to5.6, 4.5 to 5.5, 4.5 to 5.6, 4.5 to 5.5, 4.5 to 5.4, 4.5 to 5.3, 4.5 to5.2, 4.5 to 5.1, 4.5 to 5.0, 4.5 to 4.9, 4.5 to 4.8, 4.5 to 4.7, or 4.5to 4.6. In one embodiment the buffer has a pH 4.5, 4.6, 4.7, 4.8, 4.8,5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

Herein, a “surfactant” refers to a surface-active agent, preferably anonionic surfactant. Examples of surfactants herein include polysorbate(for example, polysorbate 20 and, polysorbate 80); poloxamer (e.g.poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurelsulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. In oneembodiment, the surfactant herein is polysorbate 20.

In a pharmacological sense, in the context of the invention, a“therapeutically effective amount” of an antibody refers to an amounteffective in the prevention or treatment of a disorder for the treatmentof which the antibody is effective. A “disorder” is any condition thatwould benefit from treatment with the antibody. This includes chronicand acute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question.

A “preservative” is a compound which can be optionally included in theformulation to essentially reduce bacterial action therein, thusfacilitating the production of a multi-use formulation, for example.Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkylgroups are long-chain compounds), and benzethonium chloride. Other typesof preservatives include aromatic alcohols such as phenol, butyl andbenzyl alcohol, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In oneembodiment, the preservative herein is benzyl alcohol.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. A polyol may optionally be included in the formulation. Incertain embodiments, polyols herein have a molecular weight which isless than about 600 kD (e.g. in the range from about 120 to about 400kD). A “reducing sugar” is one which contains a hemiacetal group thatcan reduce metal ions or react covalently with lysine and other aminogroups in proteins and a “nonreducing sugar” is one which does not havethese properties of a reducing sugar. Examples of reducing sugars arefructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose and glucose. Nonreducing sugars include sucrose,trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol,erythritol, threitol, sorbitol and glycerol are examples of sugaralcohols. As to sugar acids, these include L-gluconate and metallicsalts thereof. Where it desired that the formulation is freeze-thawstable, the polyol is preferably one which does not crystallize atfreezing temperatures (e.g. −20° C.) such that it destabilizes theantibody in the formulation. In certain embodiments, nonreducing sugarssuch as sucrose and trehalose are examples of polyols, with trehalosebeing preferred over sucrose, because of the solution stability oftrehalose.

The term “VEGF” or “VEGF-A” as used herein refers to the 165-amino acidhuman vascular endothelial cell growth factor and related 121-, 189-,and 206-amino acid human vascular endothelial cell growth factors, asdescribed by Leung et al. (1989) Science 246:1306, and Houck et al.(1991) Mol. Endocrin, 5:1806, together with the naturally occurringallelic and processed forms thereof. The term “VEGF” also refers toVEGFs from non-human species such as mouse, rat or primate. Sometimesthe VEGF from a specific species are indicated by terms such as hVEGFfor human VEGF, mVEGF for murine VEGF, and etc. The term “VEGF” is alsoused to refer to truncated forms of the polypeptide comprising aminoacids 8 to 109 or 1 to 109 of the 165-amino acid human vascularendothelial cell growth factor. Reference to any such forms of VEGF maybe identified in the present application, e.g., by “VEGF (8-109),” “VEGF(1-109)” or “VEGF₁₆₅.” The amino acid positions for a “truncated” nativeVEGF are numbered as indicated in the native VEGF sequence. For example,amino acid position 17 (methionine) in truncated native VEGF is alsoposition 17 (methionine) in native VEGF. The truncated native VEGF hasbinding affinity for the KDR and Flt-1 receptors comparable to nativeVEGF.

“VEGF biological activity” includes binding to any VEGF receptor or anyVEGF signaling activity such as regulation of both normal and abnormalangiogenesis and vasculogenesis (Ferrara and Davis-Smyth (1997)Endocrine Rev. 18:4-25; Ferrara (1999) J. Mol. Med. 77:527-543);promoting embryonic vasculogenesis and angiogenesis (Carmeliet et al.(1996) Nature 380:435-439; Ferrara et al. (1996) Nature 380:439-442);and modulating the cyclical blood vessel proliferation in the femalereproductive tract and for bone growth and cartilage formation (Ferraraet al. (1998) Nature Med. 4:336-340; Gerber et al. (1999) Nature Med.5:623-628). In addition to being an angiogenic factor in angiogenesisand vasculogenesis, VEGF, as a pleiotropic growth factor, exhibitsmultiple biological effects in other physiological processes, such asendothelial cell survival, vessel permeability and vasodilation,monocyte chemotaxis and calcium influx (Ferrara and Davis-Smyth (1997),supra and Cebe-Suarez et al. Cell. Mol. Life Sci. 63:601-615 (2006)).Moreover, recent studies have reported mitogenic effects of VEGF on afew non-endothelial cell types, such as retinal pigment epithelialcells, pancreatic duct cells, and Schwann cells. Guerrin et al. (1995)J. Cell Physiol. 164:385-394; Oberg-Welsh et al. (1997) Mol. Cell.Endocrinol. 126:125-132; Sondell et al. (1999) J. Neurosci.19:5731-5740.

A “VEGF antagonist” or “VEGF-specific antagonist” refers to a moleculecapable of binding to VEGF, reducing VEGF expression levels, orneutralizing, blocking, inhibiting, abrogating, reducing, or interferingwith VEGF biological activities, including, but not limited to, VEGFbinding to one or more VEGF receptors and VEGF mediated angiogenesis andendothelial cell survival or proliferation. Included as VEGF-specificantagonists useful in the methods of the invention are polypeptides thatspecifically bind to VEGF, anti-VEGF antibodies and antigen-bindingfragments thereof, receptor molecules and derivatives which bindspecifically to VEGF thereby sequestering its binding to one or morereceptors, fusions proteins (e.g., VEGF-Trap (Regeneron)), andVEGF₁₂₁-gelonin (Peregrine). VEGF-specific antagonists also includeantagonist variants of VEGF polypeptides, antisense nucleobase oligomersdirected to VEGF, small RNA molecules directed to VEGF, RNA aptamers,peptibodies, and ribozymes against VEGF. VEGF-specific antagonists alsoinclude nonpeptide small molecules that bind to VEGF and are capable ofblocking, inhibiting, abrogating, reducing, or interfering with VEGFbiological activities. Thus, the term “VEGF activities” specificallyincludes VEGF mediated biological activities of VEGF. In certainembodiments, the VEGF antagonist reduces or inhibits, by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the expression level orbiological activity of VEGF.

An “anti-VEGF antibody” is an antibody that binds to VEGF withsufficient affinity and specificity. In certain embodiments, theantibody selected will normally have a sufficiently binding affinity forVEGF, for example, the antibody may bind hVEGF with a K_(d) value ofbetween 100 nM-1 pM. Antibody affinities may be determined by a surfaceplasmon resonance based assay (such as the BIAcore assay as described inPCT Application Publication No. WO2005/012359); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), forexample.

In certain embodiment, the anti-VEGF antibody can be used as atherapeutic agent in targeting and interfering with diseases orconditions wherein the VEGF activity is involved. Also, the antibody maybe subjected to other biological activity assays, e.g., in order toevaluate its effectiveness as a therapeutic. Such assays are known inthe art and depend on the target antigen and intended use for theantibody. Examples include the HUVEC inhibition assay; tumor cell growthinhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062). An anti-VEGFantibody will usually not bind to other VEGF homologues such as VEGF-Bor VEGF-C, nor other growth factors such as P1GF, PDGF or bFGF. In oneembodiment, anti-VEGF antibody is a monoclonal antibody that binds tothe same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced byhybridoma ATCC HB 10709. In another embodiment, the anti-VEGF antibodyis a recombinant humanized anti-VEGF monoclonal antibody generatedaccording to Presta et al. (1997) Cancer Res. 57:4593-4599, includingbut not limited to the antibody known as bevacizumab (BV; AVASTIN®).

The anti-VEGF antibody “Bevacizumab (BV),” also known as “rhuMAb VEGF”or “AVASTIN®,” is a recombinant humanized anti-VEGF monoclonal antibodygenerated according to Presta et al. (1997) Cancer Res. 57:4593-4599. Itcomprises mutated human IgG1 framework regions and antigen-bindingcomplementarity-determining regions from the murine anti-hVEGFmonoclonal antibody A.4.6.1 that blocks binding of human VEGF to itsreceptors. Approximately 93% of the amino acid sequence of Bevacizumab,including most of the framework regions, is derived from human IgG1, andabout 7% of the sequence is derived from the murine antibody A4.6.1.Bevacizumab has a molecular mass of about 149,000 daltons and isglycosylated. Bevacizumab and other humanized anti-VEGF antibodies arefurther described in U.S. Pat. No. 6,884,879 issued Feb. 26, 2005, theentire disclosure of which is expressly incorporated herein byreference.

The term “B20 series polypeptide” as used herein refers to apolypeptide, including an antibody that binds to VEGF. B20 seriespolypeptides includes, but not limited to, antibodies derived from asequence of the B20 antibody or a B20-derived antibody described in USPublication No. 20060280747, US Publication No. 20070141065 and/or USPublication No. 20070020267, the content of these patent applicationsare expressly incorporated herein by reference. In one embodiment, B20series polypeptide is B20-4.1 as described in US Publication No.20060280747, US Publication No. 20070141065 and/or US Publication No.20070020267. In another embodiment, B20 series polypeptide is B20-4.1.1described in U.S. Patent Application 60/991,302, the entire disclosureof which is expressly incorporated herein by reference.

The term “G6 series polypeptide” as used herein refers to a polypeptide,including an antibody that binds to VEGF. G6 series polypeptidesincludes, but not limited to, antibodies derived from a sequence of theG6 antibody or a G6-derived antibody described in US Publication No.20060280747, US Publication No. 20070141065 and/or US Publication No.20070020267. G6 series polypeptides, as described in US Publication No.20060280747, US Publication No. 20070141065 and/or US Publication No.20070020267 include, but not limited to, G6-8, G6-23 and G6-31.

For additional antibodies see U.S. Pat. Nos. 7,060,269, 6,582,959,6,703,020; 6,054,297; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1;U.S. Patent Application Publication Nos. 2006009360, 20050186208,20030206899, 20030190317, 20030203409, and 20050112126; and Popkov etal., Journal of Immunological Methods 288:149-164 (2004). In certainembodiments, other antibodies include those that bind to a functionalepitope on human VEGF comprising of residues F17, M18, D19, Y21, Y25,Q89, I91, K101, E103, and C104 or, alternatively, comprising residuesF17, Y21, Q22, Y25, D63, I83 and Q89.

Other anti-VEGF antibodies are also known, and described, for example,in Liang et al., J Biol Chem 281, 951-961 (2006).

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

A “disorder” is any condition that would benefit from treatmentincluding, but not limited to, chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Disorders include angiogenic disorders.“Angiogenic disorder” as used herein refers to any condition involvingabnormal angiogenesis or abnormal vascular permeability or leakage.Non-limiting examples of angiogenic disorders to be treated hereininclude malignant and benign tumors; non-leukemias and lymphoidmalignancies; and, in particular, tumor (cancer) metastasis.

“Abnormal angiogenesis” occurs when new blood vessels grow eitherexcessively or otherwise inappropriately (e.g., the location, timing,degree, or onset of the angiogenesis being undesired from a medicalstandpoint) in a diseased state or such that it causes a diseased state.In some cases, excessive, uncontrolled, or otherwise inappropriateangiogenesis occurs when there is new blood vessel growth thatcontributes to the worsening of the diseased state or cause of adiseased state. The new blood vessels can feed the diseased tissues,destroy normal tissues, and in the case of cancer, the new vessels canallow tumor cells to escape into the circulation and lodge in otherorgans (tumor metastases). Examples of disorders involving abnormalangiogenesis include, but are not limited to cancer, especiallyvascularized solid tumors and metastatic tumors (including colon, lungcancer (especially small-cell lung cancer), or prostate cancer),diseases caused by ocular neovascularisation, especially diabeticblindness, retinopathies, primarily diabetic retinopathy or age-relatedmacular degeneration, choroidal neovascularization (CNV), diabeticmacular edema, pathological myopia, von Hippel-Lindau disease,histoplasmosis of the eye, Central Retinal Vein Occlusion (CRVO),corneal neovascularization, retinal neovascularization and rubeosis;psoriasis, psoriatic arthritis, haemangioblastoma such as haemangioma;inflammatory renal diseases, such as glomerulonephritis, especiallymesangioproliferative glomerulonephritis, haemolytic uremic syndrome,diabetic nephropathy or hypertensive nephrosclerosis; variousimflammatory diseases, such as arthritis, especially rheumatoidarthritis, inflammatory bowel disease, psoriasis, sarcoidosis, arterialarteriosclerosis and diseases occurring after transplants, endometriosisor chronic asthma and other conditions.

“Abnormal vascular permeability” occurs when the flow of fluids,molecules (e.g., ions and nutrients) and cells (e.g., lymphocytes)between the vascular and extravascular compartments is excessive orotherwise inappropriate (e.g., the location, timing, degree, or onset ofthe vascular permeability being undesired from a medical standpoint) ina diseased state or such that it causes a diseased state. Abnormalvascular permeability may lead to excessive or otherwise inappropriate“leakage” of ions, water, nutrients, or cells through the vasculature.In some cases, excessive, uncontrolled, or otherwise inappropriatevascular permeability or vascular leakage exacerbates or induces diseasestates including, e.g., edema associated with tumors including, e.g.,brain tumors; ascites associated with malignancies; Meigs' syndrome;lung inflammation; nephrotic syndrome; pericardial effusion; pleuraleffusion; permeability associated with cardiovascular diseases such asthe condition following myocardial infarctions and strokes and the like.The present invention contemplates treating those patients that havedeveloped or are at risk of developing the diseases and disordersassociated with abnormal vascular permeability or leakage.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer. In one embodiment, the cell proliferative disorder is a tumor.

“Tumor,” as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include, but notlimited to, squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer and gastrointestinalstromal cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, cancer of the urinarytract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma,superficial spreading melanoma, lentigo maligna melanoma, acrallentiginous melanomas, nodular melanomas, multiple myeloma and B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), Meigs' syndrome,brain, as well as head and neck cancer, and associated metastases. Incertain embodiments, cancers that are amenable to treatment by theantibodies of the invention include breast cancer, colorectal cancer,rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkinslymphoma (NHL), renal cell cancer, prostate cancer, liver cancer,pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoidcarcinoma, head and neck cancer, ovarian cancer, mesothelioma, andmultiple myeloma. In some embodiments, the cancer is selected from:small cell lung cancer, gliblastoma, neuroblastomas, melanoma, breastcarcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellularcarcinoma. Yet, in some embodiments, the cancer is selected from:non-small cell lung cancer, colorectal cancer, glioblastoma and breastcarcinoma, including metastatic forms of those cancers.

The term “anti-cancer therapy” refers to a therapy useful in treatingcancer. Examples of anti-cancer therapeutic agents include, but arelimited to, e.g., chemotherapeutic agents, growth inhibitory agents,cytotoxic agents, agents used in radiation therapy, anti-angiogenicagents, apoptotic agents, anti-tubulin agents, and other agents to treatcancer, such as anti-HER-2 antibodies, anti-CD20 antibodies, anepidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosinekinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib (Tarceva™),platelet derived growth factor inhibitors (e.g., Gleevec™ (ImatinibMesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, cytokines,antagonists (e.g., neutralizing antibodies) that bind to one or more ofthe following targets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMAor VEGF receptor(s), TRAIL/Apo2, and other bioactive and organicchemical agents, etc. Combinations thereof are also included in theinvention.

An “angiogenic factor or agent” is a growth factor or its receptor whichis involved in stimulating the development of blood vessels, e.g.,promote angiogenesis, endothelial cell growth, stability of bloodvessels, and/or vasculogenesis, etc. For example, angiogenic factors,include, but are not limited to, e.g., VEGF and members of the VEGFfamily and their receptors (VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2 andVEGFR3), P1GF, PDGF family, fibroblast growth factor family (FGFs), TIEligands (Angiopoietins, ANGPT1, ANGPT2), TIE1, TIE2, ephrins, Bv8,Delta-like ligand 4 (DLL4), Del-1, fibroblast growth factors: acidic(aFGF) and basic (bFGF), FGF4, FGF9, BMP9, BMP10, Follistatin,Granulocyte colony-stimulating factor (G-CSF), GM-CSF, Hepatocyte growthfactor (HGF)/scatter factor (SF), Interleukin-8 (IL-8), CXCL12, Leptin,Midkine, neuropilins, NRP1, NRP2, Placental growth factor,Platelet-derived endothelial cell growth factor (PD-ECGF),Platelet-derived growth factor, especially PDGF-BB, PDGFR-alpha, orPDGFR-beta, Pleiotrophin (PTN), Progranulin, Proliferin, Transforminggrowth factor-alpha (TGF-alpha), Transforming growth factor-beta(TGF-beta), Tumor necrosis factor-alpha (TNF-alpha), Alk1, CXCR4,Notch1, Notch4, Sema3A, Sema3C, Sema3F, Robo4, etc. It would furtherinclude factors that promote angiogenesis, such as ESM1 and Perlecan. Itwould also include factors that accelerate wound healing, such as growthhormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growthfactor (EGF), EGF-like domain, multiple 7 (EGFL7), CTGF and members ofits family, and TGF-alpha and TGF-beta. See, e.g., Klagsbrun and D'Amore(1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene22:3172-3179; Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364;Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 1 listing knownangiogenic factors); and, Sato (2003) Int. J. Clin. Oncol. 8:200-206.

An “anti-angiogenic agent” or “angiogenic inhibitor” refers to a smallmolecular weight substance, a polynucleotide (including, e.g., aninhibitory RNA (RNAi or siRNA)), a polypeptide, an isolated protein, arecombinant protein, an antibody, or conjugates or fusion proteinsthereof, that inhibits angiogenesis, vasculogenesis, or undesirablevascular permeability, either directly or indirectly. It should beunderstood that the anti-angiogenic agent includes those agents thatbind and block the angiogenic activity of the angiogenic factor or itsreceptor. For example, an anti-angiogenic agent is an antibody or otherantagonist to an angiogenic agent as defined above, e.g., antibodies toVEGF-A or to the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor),anti-PDGFR inhibitors, small molecules that block VEGF receptorsignaling (e.g., PTK787/ZK2284, SU6668, SUTENT®/SU11248 (sunitinibmalate), AMG706, or those described in, e.g., international patentapplication WO 2004/113304). Anti-angiogenic agents include, but are notlimited to, the following agents: VEGF inhibitors such as aVEGF-specific antagonist, EGF inhibitor, EGFR inhibitors, Erbitux®(cetuximab, ImClone Systems, Inc., Branchburg, N.J.), Vectibix®(panitumumab, Amgen, Thousand Oaks, Calif.), TIE2 inhibitors, IGF1Rinhibitors, COX-II (cyclooxygenase II) inhibitors, MMP-2(matrix-metalloproteinase 2) inhibitors, and MMP-9(matrix-metalloproteinase 9) inhibitors, CP-547,632 (Pfizer Inc., NY,USA), Axitinib (Pfizer Inc.; AG-013736), ZD-6474 (AstraZeneca), AEE788(Novartis), AZD-2171), VEGF Trap (Regeneron/Aventis), Vatalanib (alsoknown as PTK-787, ZK-222584: Novartis & Schering A G), Macugen(pegaptanib octasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech),IM862 (Cytran Inc. of Kirkland, Wash., USA); and angiozyme, a syntheticribozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.)and combinations thereof. Other angiogenesis inhibitors includethrombospondin1, thrombospondin2, collagen IV and collagen XVIII. VEGFinhibitors are disclosed in U.S. Pat. Nos. 6,534,524 and 6,235,764, bothof which are incorporated in their entirety for all purposes.Anti-angiogenic agents also include native angiogenesis inhibitors,e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore(1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar (2003) Oncogene22:3172-3179 (e.g., Table 3 listing anti-angiogenic therapy in malignantmelanoma); Ferrara & Alitalo (1999) Nature Medicine 5(12):1359-1364;Tonini et al. (2003) Oncogene 22:6549-6556 (e.g., Table 2 listing knownantiangiogenic factors); and, Sato (2003) Int. J. Clin. Oncol. 8:200-206(e.g., Table 1 listing anti-angiogenic agents used in clinical trials).

The term “anti-angiogenic therapy” refers to a therapy useful forinhibiting angiogenesis which comprises the administration of ananti-angiogenic agent.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu), chemotherapeutic agents (e.g.,methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine,etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil,daunorubicin or other intercalating agents, enzymes and fragmentsthereof such as nucleolytic enzymes, antibiotics, and toxins such assmall molecule toxins or enzymatically active toxins of bacterial,fungal, plant or animal origin, including fragments and/or variantsthereof, and the various antitumor or anticancer agents disclosed below.Other cytotoxic agents are described below. A tumoricidal agent causesdestruction of tumor cells.

A “toxin” is any substance capable of having a detrimental effect on thegrowth or proliferation of a cell.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®);alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; a camptothecin (including the synthetic analogue topotecan(HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin,scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); podophyllotoxin; podophyllinic acid; teniposide;cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaIl (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin,including dynemicin A; an esperamicin; as well as neocarzinostatinchromophore and related chromoprotein enediyne antibiotic chromophores),aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins,dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HClliposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®),peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin),epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such asmitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil(5-FU); combretastatin; folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2′-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoid, e.g., paclitaxel (TAXOL®,Bristol-Myers Squibb Oncology, Princeton, N.J.), albumin-engineerednanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel(TAXOTERE®, Rhôme-Poulene Rorer, Antony, France); chloranbucil;6-thioguanine; mercaptopurine; methotrexate; platinum agents such ascisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, whichprevent tubulin polymerization from forming microtubules, includingvinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®,FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide;mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin;aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid,including bexarotene (TARGRETIN®); bisphosphonates such as clodronate(for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095,zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®),pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®);troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisenseoligonucleotides, particularly those that inhibit expression of genes insignaling pathways implicated in aberrant cell proliferation, such as,for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R) (e.g., erlotinib (Tarceva™)); and VEGF-A that reduce cellproliferation; vaccines such as THERATOPE® vaccine and gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH(e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib,SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib oretoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®);CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors; tyrosinekinase inhibitors; serine-threonine kinase inhibitors such as rapamycin(sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such aslonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts,acids or derivatives of any of the above; as well as combinations of twoor more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone;and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin, and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

Chemotherapeutic agents as defined herein include “anti-hormonal agents”or “endocrine therapeutics” which act to regulate, reduce, block, orinhibit the effects of hormones that can promote the growth of cancer.They may be hormones themselves, including, but not limited to:anti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON•toremifene; aromatase inhibitorsthat inhibit the enzyme aromatase, which regulates estrogen productionin the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane,formestanie, fadrozole, RIVISOR® vorozole, FEMARA® letrozole, andARIMIDEX® anastrozole; and anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides,particularly those which inhibit expression of genes in signalingpathways implicated in abherant cell proliferation, such as, forexample, PKC-alpha, Raf and H-Ras; ribozymes such as a VEGF expressioninhibitor (e.g., ANGIOZYME® ribozyme) and a HER2 expression inhibitor;vaccines such as gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2;LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; Vinorelbine andEsperamicins (see U.S. Pat. No. 4,675,187), and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.In one embodiment, growth inhibitory agent is growth inhibitory antibodythat prevents or reduces proliferation of a cell expressing an antigento which the antibody binds. In another embodiment, the growthinhibitory agent may be one which significantly reduces the percentageof cells in S phase. Examples of growth inhibitory agents include agentsthat block cell cycle progression (at a place other than S phase), suchas agents that induce G1 arrest and M-phase arrest. Classical M-phaseblockers include the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in Mendelsohn and Israel, eds., The Molecular Basis of Cancer,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W.B. Saunders, Philadelphia,1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) areanticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®,Rhone-Poulenc Rorer), derived from the European yew, is a semisyntheticanalogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel anddocetaxel promote the assembly of microtubules from tubulin dimers andstabilize microtubules by preventing depolymerization, which results inthe inhibition of mitosis in cells.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “constant domain” refers to the portion of an immunoglobulinmolecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable domain, which containsthe antigen binding site. The constant domain contains the C_(H)1,C_(H)2 and C_(H)3 domains (collectively, CH) of the heavy chain and theCHL (or CL) domain of the light chain.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “VH.” Thevariable domain of the light chain may be referred to as “VL.” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (κ) and lambda (λ), based on the amino acid sequences of theirconstant domains.

The term IgG “isotype: or “subclass” as used herein is meant any of thesubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)₂ antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal., Nature Biotechnol. 14: 845-851 (1996); Neuberger, NatureBiotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g.,U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATTZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen(e.g., has a binding affinity (Kd) value of no more than about 1×10-7 M,preferably no more than about 1×10-8 M and preferably no more than about1×10-9 M) but has a binding affinity for a homologue of the antigen froma second nonhuman mammalian species which is at least about 50 fold, orat least about 500 fold, or at least about 1000 fold, weaker than itsbinding affinity for the human antigen. The species-dependent antibodycan be any of the various types of antibodies as defined above, butpreferably is a humanized or human antibody.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g, Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

As used herein, “library” refers to a plurality of antibody or antibodyfragment sequences (for example, polypeptides of the invention), or thenucleic acids that encode these sequences, the sequences being differentin the combination of variant amino acids that are introduced into thesesequences according to the methods of the invention.

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target antigen with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman (1992) Curr. Opin.Struct. Biol. 3:355-362, and references cited therein. In a monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells (1991) Methods: A companion to Methodsin Enzymology 3:205-0216.

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. The phagemid may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage. Theplasmid will also generally contain a selectable marker for antibioticresistance. Segments of DNA cloned into these vectors can be propagatedas plasmids. When cells harboring these vectors are provided with allgenes necessary for the production of phage particles, the mode ofreplication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

II. Modes for Carrying Out the Invention

The invention herein relates to a stable aqueous formulation comprisingan antibody. The antibody in the formulation is prepared usingtechniques available in the art for generating antibodies, exemplarymethods of which are described in more detail in the following sections.

The antibody is directed against an antigen of interest. Preferably, theantigen is a biologically important polypeptide and administration ofthe antibody to a mammal suffering from a disorder can result in atherapeutic benefit in that mammal. However, antibodies directed againstnonpolypeptide antigens are also contemplated.

Where the antigen is a polypeptide, it may be a transmembrane molecule(e.g. receptor) or ligand such as a growth factor. Exemplary antigensinclude molecules such as vascular endothelial growth factor (VEGF);ox-LDL; ox-ApoB100; renin; a growth hormone, including human growthhormone and bovine growth hormone; growth hormone releasing factor;parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tssue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tssue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; enkephalinase; RANTES (regulated on activationnormally T-cell expressed and secreted); human macrophage inflammatoryprotein (MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; receptors for hormonesor growth factors; protein A or D; rheumatoid factors; a neurotrophicfactor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor suchas NGF-β; platelet-derived growth factor (PDGF); fibroblast growthfactor such as aFGF and bFGF; epidermal growth factor (EGF);transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4, or TGF-β 5; insulin-likegrowth factor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I (brainIGF-I), insulin-like growth factor binding proteins; CD proteins such asCD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs),e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10;superoxide dismutase; T-cell receptors; surface membrane proteins; decayaccelerating factor; viral antigen such as, for example, a portion ofthe AIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; integrns such as CD11a, CD11b, CD11c, CD18, anICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 orHER4 receptor; and fragments of any of the above-listed polypeptides.

In certain embodiments of the invention, the molecular targets forantibodies encompassed by the invention include VEGF. In one embodiment,the antibody herein is one which binds to human VEGF.

A. Preparation of the Formulation

After preparation of the antibody of interest (e.g., techniques forproducing antibodies which can be formulated as disclosed herein will beelaborated below and are known in the art), the pharmaceuticalformulation comprising it is prepared. In certain embodiments, theantibody to be formulated has not been subjected to prior lyophilizationand the formulation of interest herein is an aqueous formulation. Incertain embodiments, the antibody is a full length antibody. In oneembodiment, the antibody in the formulation is an antibody fragment,such as an F(ab′)₂, in which case problems that may not occur for thefull length antibody (such as clipping of the antibody to Fab) may needto be addressed. The therapeutically effective amount of antibodypresent in the formulation is determined by taking into account thedesired dose volumes and mode(s) of administration, for example. Fromabout 0.1 mg/mL to about 250 mg/mL, or from about 10 mg/mL to about 200mg/mL or from about 50 mg/mL to about 175 mg/mL is an exemplary antibodyconcentration in the formulation.

An aqueous formulation is prepared comprising the antibody in apH-buffered solution. The buffer of this invention has a pH in the rangefrom about 4.0 to about 6.5. In certain embodiments the pH is in therange from pH 4.25 to 6.25, or in the range from pH 4.5 to 6.0, or inthe range from pH 4.75 to 5.75, or in the range from pH 5.0 to 5.5, orin the range from pH 5.1 to 5.4. In certain embodiments of theinvention, the formulation has a pH of 5.2 or about 5.2. Examples ofbuffers that will control the pH within this range include acetate (e.g.arginine acetate or sodium acetate), succinate (such as argininesuccinate or sodium succinate), gluconate, citrate and other organicacid buffers and combinations thereof. The buffer concentration can befrom about 1 mM to about 600 mM, depending, for example, on the bufferand the desired isotonicity of the formulation. In certain embodiments,the buffer contains arginine in the concentration of 50 mM to 500 mM, 75mM to 400 mM, 100 mM to 250 mM, 120 mM to 240 mM, 150 mM to 225 mM, or175 mM to 210 mM. In certain embodiments of the invention, the buffercontains arginine in the concentration of 200 mM or about 200 mM. In oneembodiment, the buffer is arginine acetate (e.g., at 200 mM or about 200mM), pH 5.2.

A surfactant can optionally be added to the antibody formulation.Exemplary surfactants include nonionic surfactants such as polysorbates(e.g. polysorbates 20, 80 etc) or poloxamers (e.g. poloxamer 188). Theamount of surfactant added is such that it reduces aggregation of theformulated antibody and/or minimizes the formation of particulates inthe formulation and/or reduces adsorption. For example, the surfactantmay be present in the formulation in an amount from about 0.001% toabout 0.5%, from about 0.005% to about 0.2%, from about 0.01% to about0.1%, or from about 0.02% to about 0.06%, or about 0.03% to about 0.05%.In certain embodiments, the surfactant is present in the formulation inan amount of 0.04% or about 0.04%. In one embodiment, the formulationdoes not comprise a surfactant.

In one embodiment, the formulation contains the above-identified agents(e.g., antibody, buffer, and/or surfactant) and is essentially free ofone or more preservatives, such as benzyl alcohol, phenol, m-cresol,chlorobutanol and benzethonium Cl. In another embodiment, a preservativemay be included in the formulation, particularly where the formulationis a multidose formulation. The concentration of preservative may be inthe range from about 0.1% to about 2%, preferably from about 0.5% toabout 1%. One or more other pharmaceutically acceptable carriers,excipients or stabilizers such as those described in Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980) may beincluded in the formulation provided that they do not adversely affectthe desired characteristics of the formulation. Acceptable carriers,excipients or stabilizers are nontoxic to recipients at the dosages andconcentrations employed and include; additional buffering agents;co-solvents; anti-oxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions. Exemplary pharmaceutically acceptable carriersherein further include insterstitial drug dispersion agents such assoluble neutral-active hyaluronidase glycoproteins (sHASEGP), forexample, human soluble PH-20 hyaluronidase glycoproteins, such asrHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplarysHASEGPs and methods of use, including rHuPH20, are described in USPatent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, asHASEGP is combined with one or more additional glycosaminoglycanasessuch as chondroitinases.

While the various descriptions of chelators herein often focus on EDTA,it will be appreciated that other metal ion chelators are alsoencompassed within the invention. Metal ion chelators are well known bythose of skill in the art and include, but are not necessarily limitedto aminopolycarboxylates, EDTA (ethylenediaminetetraacetic acid), EGTA(ethylene glycol-bis(beta-aminoethyl ether)-N,N,N′,N′-tetraacetic acid),NTA (nitrilotriacetic acid), EDDS (ethylene diamine disuccinate), PDTA(1,3-propylenediaminetetraacetic acid), DTPA(diethylenetriaminepentaacetic acid), ADA (beta-alaninediacetic acid),MGCA (methylglycinediacetic acid), etc. Additionally, some embodimentsherein comprise phosphonates/phosphonic acid chelators.

The formulation herein may also contain more than one protein asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect the otherprotein. For example, where the antibody is anti-VEGF, it may becombined with another agent (e.g., a chemotherapeutic agent, andanti-neoplastic agent, and anti.

The formulations to be used for in vivo administration should besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to, or following, preparation of theformulation.

B. Administration of the Formulation

The formulation is administered to a mammal in need of treatment withthe antibody, preferably a human, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. In one embodiment, the formulation isadministered to the mammal by intravenous administration. For suchpurposes, the formulation may be injected using a syringe or via an IVline, for example. In one embodiment, the formulation is administered tothe mammal by subcutaneous administration.

The appropriate dosage (“therapeutically effective amount”) of theantibody will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, the type ofantibody used, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments and may be administered to the patient at any timefrom diagnosis onwards. The antibody may be administered as the soletreatment or in conjunction with other drugs or therapies useful intreating the condition in question.

As a general proposition, the therapeutically effective amount of theantibody administered will be in the range of about 0.1 to about 50mg/kg of patent body weight whether by one or more administrations, withthe typical range of antibody used being about 0.3 to about 20 mg/kg,preferably about 0.3 to about 15 mg/kg, administered daily, for example.However, other dosage regimens may be useful. In one embodiment, theantagonist is an anti-VEGF antibody that is administered at a dose ofabout 100 or 400 mg every 1, 2, 3, or 4 weeks or is administered a doseof about 1, 3, 5, 7.5, 10, 15, or 20 mg/kg every 1, 2, 3, or 4 weeks.The dose may be administered as a single dose or as multiple doses(e.g., 2 or 3 doses), such as infusions. The progress of this therapy iseasily monitored by conventional techniques.

C. Antibody Preparation

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Certain Antibody-Based Methods

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies of the invention can be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), andfurther described, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260(1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) regardinghuman-human hybridomas. Additional methods include those described, forexample, in U.S. Pat. No. 7,189,826 regarding production of monoclonalhuman natural IgM antibodies from hybridoma cell lines. Human hybridomatechnology (Trioma technology) is described in Vollmers and Brandlein,Histology and Histopathology, 20(3):927-937 (2005) and Vollmers andBrandlein, Methods and Findings in Experimental and ClinicalPharmacology, 27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide of the invention or a fragment thereof,and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton,Mont.). A polypeptide of the invention (e.g., antigen) or a fragmentthereof may be prepared using methods well known in the art, such asrecombinant methods, some of which are further described herein. Serumfrom immunized animals is assayed for anti-antigen antibodies, andbooster immunizations are optionally administered. Lymphocytes fromanimals producing anti-antigen antibodies are isolated. Alternatively,lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to an antibody of theinvention. The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbent assay (ELISA). The binding affinity of the monoclonalantibody can be determined, for example, by Scatchard analysis. See,e.g., Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

(iii) Certain Library Screening Methods

Antibodies of the invention can be made by using combinatorial librariesto screen for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are describedgenerally in Hoogenboom et al. in Methods in Molecular Biology 178:1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001). For example, onemethod of generating antibodies of interest is through the use of aphage antibody library as described in Lee et al., J. Mol. Biol. (2004),340(5):1073-93.

In principle, synthetic antibody clones are selected by screening phagelibraries containing phage that display various fragments of antibodyvariable region (Fv) fused to phage coat protein. Such phage librariesare panned by affinity chromatography against the desired antigen.Clones expressing Fv fragments capable of binding to the desired antigenare adsorbed to the antigen and thus separated from the non-bindingclones in the library. The binding clones are then eluted from theantigen, and can be further enriched by additional cycles of antigenadsorption/elution. Any of the antibodies of the invention can beobtained by designing a suitable antigen screening procedure to selectfor the phage clone of interest followed by construction of a fulllength antibody clone using the Fv sequences from the phage clone ofinterest and suitable constant region (Fc) sequences described in Kabatet al., Sequences of Proteins of Immunological Interest, Fifth Edition,NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.

In certain embodiments, the antigen-binding domain of an antibody isformed from two variable (V) regions of about 110 amino acids, one eachfrom the light (VL) and heavy (VH) chains, that both present threehypervariable loops (HVRs) or complementarity-determining regions(CDRs). Variable domains can be displayed functionally on phage, eitheras single-chain Fv (scFv) fragments, in which VH and VL are covalentlylinked through a short, flexible peptide, or as Fab fragments, in whichthey are each fused to a constant domain and interact non-covalently, asdescribed in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Asused herein, scFv encoding phage clones and Fab encoding phage clonesare collectively referred to as “Fv phage clones” or “Fv clones.”

Repertoires of VH and VL genes can be separately cloned by polymerasechain reaction (PCR) and recombined randomly in phage libraries, whichcan then be searched for antigen-binding clones as described in Winteret al., Ann. Rev. Immunol., 12: 433-455 (1994). Libraries from immunizedsources provide high-affinity antibodies to the immunogen without therequirement of constructing hybridomas. Alternatively, the naiverepertoire can be cloned to provide a single source of human antibodiesto a wide range of non-self and also self antigens without anyimmunization as described by Griffiths et al., EMBO J, 12: 725-734(1993). Finally, naive libraries can also be made synthetically bycloning the unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

In certain embodiments, filamentous phage is used to display antibodyfragments by fusion to the minor coat protein pIII. The antibodyfragments can be displayed as single chain Fv fragments, in which VH andVL domains are connected on the same polypeptide chain by a flexiblepolypeptide spacer, e.g. as described by Marks et al., J. Mol. Biol.,222: 581-597 (1991), or as Fab fragments, in which one chain is fused topIII and the other is secreted into the bacterial host cell periplasmwhere assembly of a Fab-coat protein structure which becomes displayedon the phage surface by displacing some of the wild type coat proteins,e.g. as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137(1991).

In general, nucleic acids encoding antibody gene fragments are obtainedfrom immune cells harvested from humans or animals. If a library biasedin favor of anti-antigen clones is desired, the subject is immunizedwith antigen to generate an antibody response, and spleen cells and/orcirculating B cells other peripheral blood lymphocytes (PBLs) arerecovered for library construction. In one embodiment, a human antibodygene fragment library biased in favor of anti-antigen clones is obtainedby generating an anti-antigen antibody response in transgenic micecarrying a functional human immunoglobulin gene array (and lacking afunctional endogenous antibody production system) such that antigenimmunization gives rise to B cells producing human antibodies againstantigen. The generation of human antibody-producing transgenic mice isdescribed below.

Additional enrichment for anti-antigen reactive cell populations can beobtained by using a suitable screening procedure to isolate B cellsexpressing antigen-specific membrane bound antibody, e.g., by cellseparation using antigen affinity chromatography or adsorption of cellsto fluorochrome-labeled antigen followed by flow-activated cell sorting(FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs froman unimmunized donor provides a better representation of the possibleantibody repertoire, and also permits the construction of an antibodylibrary using any animal (human or non-human) species in which antigenis not antigenic. For libraries incorporating in vitro antibody geneconstruction, stem cells are harvested from the subject to providenucleic acids encoding unrearranged antibody gene segments. The immunecells of interest can be obtained from a variety of animal species, suchas human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,bovine, equine, and avian species, etc.

Nucleic acid encoding antibody variable gene segments (including VH andVL segments) are recovered from the cells of interest and amplified. Inthe case of rearranged VH and VL gene libraries, the desired DNA can beobtained by isolating genomic DNA or mRNA from lymphocytes followed bypolymerase chain reaction (PCR) with primers matching the 5′ and 3′ endsof rearranged VH and VL genes as described in Orlandi et al., Proc.Natl. Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse Vgene repertoires for expression. The V genes can be amplified from cDNAand genomic DNA, with back primers at the 5′ end of the exon encodingthe mature V-domain and forward primers based within the J-segment asdescribed in Orlandi et al. (1989) and in Ward et al., Nature, 341:544-546 (1989). However, for amplifying from cDNA, back primers can alsobe based in the leader exon as described in Jones et al., Biotechnol.,9: 88-89 (1991), and forward primers within the constant region asdescribed in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732(1989). To maximize complementarity, degeneracy can be incorporated inthe primers as described in Orlandi et al. (1989) or Sastry et al.(1989). In certain embodiments, library diversity is maximized by usingPCR primers targeted to each V-gene family in order to amplify allavailable VH and VL arrangements present in the immune cell nucleic acidsample, e.g. as described in the method of Marks et al., J. Mol. Biol.,222: 581-597 (1991) or as described in the method of Orum et al.,Nucleic Acids Res., 21: 4491-4498 (1993). For cloning of the amplifiedDNA into expression vectors, rare restriction sites can be introducedwithin the PCR primer as a tag at one end as described in Orlandi et al.(1989), or by further PCR amplification with a tagged primer asdescribed in Clackson et al., Nature, 352: 624-628 (1991).

Repertoires of synthetically rearranged V genes can be derived in vitrofrom V gene segments. Most of the human VH-gene segments have beencloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,3: 88-94 (1993); these cloned segments (including all the majorconformations of the H1 and H2 loop) can be used to generate diverse VHgene repertoires with PCR primers encoding H3 loops of diverse sequenceand length as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be made with all the sequencediversity focused in a long H3 loop of a single length as described inBarbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). HumanVκ and Vλ segments have been cloned and sequenced (reported in Williamsand Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can be used tomake synthetic light chain repertoires. Synthetic V gene repertoires,based on a range of VH and VL folds, and L3 and H3 lengths, will encodeantibodies of considerable structural diversity. Following amplificationof V-gene encoding DNAs, germline V-gene segments can be rearranged invitro according to the methods of Hoogenboom and Winter, J. Mol. Biol.,227: 381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH andVL gene repertoires together in several ways. Each repertoire can becreated in different vectors, and the vectors recombined in vitro, e.g.,as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo bycombinatorial infection, e.g., the loxP system described in Waterhouseet al., Nucl. Acids Res., 21: 2265-2266 (1993). The in vivorecombination approach exploits the two-chain nature of Fab fragments toovercome the limit on library size imposed by E. coli transformationefficiency. Naive VH and VL repertoires are cloned separately, one intoa phagemid and the other into a phage vector. The two libraries are thencombined by phage infection of phagemid-containing bacteria so that eachcell contains a different combination and the library size is limitedonly by the number of cells present (about 10¹² clones). Both vectorscontain in vivo recombination signals so that the VH and VL genes arerecombined onto a single replicon and are co-packaged into phagevirions. These huge libraries provide large numbers of diverseantibodies of good affinity (K_(d) ⁻¹ of about 10⁻⁸ M).

Alternatively, the repertoires may be cloned sequentially into the samevector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci. USA,88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g.as described in Clackson et al., Nature, 352: 624-628 (1991). PCRassembly can also be used to join VH and VL DNAs with DNA encoding aflexible peptide spacer to form single chain Fv (scFv) repertoires. Inyet another technique, “in cell PCR assembly” is used to combine VH andVL genes within lymphocytes by PCR and then clone repertoires of linkedgenes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837(1992).

The antibodies produced by naive libraries (either natural or synthetic)can be of moderate affinity (K_(d) ⁻¹ of about 10⁶ to 10⁷ M⁻¹), butaffinity maturation can also be mimicked in vitro by constructing andreselecting from secondary libraries as described in Winter et al.(1994), supra. For example, mutation can be introduced at random invitro by using error-prone polymerase (reported in Leung et al.,Technique, 1: 11-15 (1989)) in the method of Hawkins et al., J. Mol.Biol., 226: 889-896 (1992) or in the method of Gram et al., Proc. Natl.Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity maturationcan be performed by randomly mutating one or more CDRs, e.g. using PCRwith primers carrying random sequence spanning the CDR of interest, inselected individual Fv clones and screening for higher affinity clones.WO 9607754 (published 14 Mar. 1996) described a method for inducingmutagenesis in a complementarity determining region of an immunoglobulinlight chain to create a library of light chain genes. Another effectiveapproach is to recombine the VH or VL domains selected by phage displaywith repertoires of naturally occurring V domain variants obtained fromunimmunized donors and screen for higher affinity in several rounds ofchain reshuffling as described in Marks et al., Biotechnol., 10: 779-783(1992). This technique allows the production of antibodies and antibodyfragments with affinities of about 10⁻⁹ M or less.

Screening of the libraries can be accomplished by various techniquesknown in the art. For example, antigen can be used to coat the wells ofadsorption plates, expressed on host cells affixed to adsorption platesor used in cell sorting, or conjugated to biotin for capture withstreptavidin-coated beads, or used in any other method for panning phagedisplay libraries.

The phage library samples are contacted with immobilized antigen underconditions suitable for binding at least a portion of the phageparticles with the adsorbent. Normally, the conditions, including pH,ionic strength, temperature and the like are selected to mimicphysiological conditions. The phages bound to the solid phase are washedand then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described inMarks et al., J. Mol. Biol., 222: 581-597 (1991), or by antigencompetition, e.g. in a procedure similar to the antigen competitionmethod of Clackson et al., Nature, 352: 624-628 (1991). Phages can beenriched 20-1,000-fold in a single round of selection. Moreover, theenriched phages can be grown in bacterial culture and subjected tofurther rounds of selection.

The efficiency of selection depends on many factors, including thekinetics of dissociation during washing, and whether multiple antibodyfragments on a single phage can simultaneously engage with antigen.Antibodies with fast dissociation kinetics (and weak binding affinities)can be retained by use of short washes, multivalent phage display andhigh coating density of antigen in solid phase. The high density notonly stabilizes the phage through multivalent interactions, but favorsrebinding of phage that has dissociated. The selection of antibodieswith slow dissociation kinetics (and good binding affinities) can bepromoted by use of long washes and monovalent phage display as describedin Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and alow coating density of antigen as described in Marks et al.,Biotechnol., 10: 779-783 (1992).

It is possible to select between phage antibodies of differentaffinities, even with affinities that differ slightly, for antigen.However, random mutation of a selected antibody (e.g. as performed insome affinity maturation techniques) is likely to give rise to manymutants, most binding to antigen, and a few with higher affinity. Withlimiting antigen, rare high affinity phage could be competed out. Toretain all higher affinity mutants, phages can be incubated with excessbiotinylated antigen, but with the biotinylated antigen at aconcentration of lower molarity than the target molar affinity constantfor antigen. The high affinity-binding phages can then be captured bystreptavidin-coated paramagnetic beads. Such “equilibrium capture”allows the antibodies to be selected according to their affinities ofbinding, with sensitivity that permits isolation of mutant clones withas little as two-fold higher affinity from a great excess of phages withlower affinity. Conditions used in washing phages bound to a solid phasecan also be manipulated to discriminate on the basis of dissociationkinetics.

Anti-antigen clones may be selected based on activity. In certainembodiments, the invention provides anti-antigen antibodies that bind toliving cells that naturally express antigen or bind to free floatingantigen or antigen attached to other cellular structures. Fv clonescorresponding to such anti-antigen antibodies can be selected by (1)isolating anti-antigen clones from a phage library as described above,and optionally amplifying the isolated population of phage clones bygrowing up the population in a suitable bacterial host; (2) selectingantigen and a second protein against which blocking and non-blockingactivity, respectively, is desired; (3) adsorbing the anti-antigen phageclones to immobilized antigen; (4) using an excess of the second proteinto elute any undesired clones that recognize antigen-bindingdeterminants which overlap or are shared with the binding determinantsof the second protein; and (5) eluting the clones which remain adsorbedfollowing step (4). Optionally, clones with the desiredblocking/non-blocking properties can be further enriched by repeatingthe selection procedures described herein one or more times.

DNA encoding hybridoma-derived monoclonal antibodies or phage display Fvclones of the invention is readily isolated and sequenced usingconventional procedures (e.g. by using oligonucleotide primers designedto specifically amplify the heavy and light chain coding regions ofinterest from hybridoma or phage DNA template). Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of the desiredmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of antibody-encoding DNA includeSkerra et al., Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun,Immunol. Revs, 130: 151 (1992).

DNA encoding the Fv clones of the invention can be combined with knownDNA sequences encoding heavy chain and/or light chain constant regions(e.g. the appropriate DNA sequences can be obtained from Kabat et al.,supra) to form clones encoding full or partial length heavy and/or lightchains. It will be appreciated that constant regions of any isotype canbe used for this purpose, including IgG, IgM, IgA, IgD, and IgE constantregions, and that such constant regions can be obtained from any humanor animal species. An Fv clone derived from the variable domain DNA ofone animal (such as human) species and then fused to constant region DNAof another animal species to form coding sequence(s) for “hybrid,” fulllength heavy chain and/or light chain is included in the definition of“chimeric” and “hybrid” antibody as used herein. In certain embodiments,an Fv clone derived from human variable DNA is fused to human constantregion DNA to form coding sequence(s) for full- or partial-length humanheavy and/or light chains.

DNA encoding anti-antigen antibody derived from a hybridoma of theinvention can also be modified, or example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofhomologous murine sequences derived from the hybridoma clone (e.g. as inthe method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855(1984)). DNA encoding a hybridoma- or Fv clone-derived antibody orfragment can be further modified by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid”antibodies are prepared that have the binding specificity of the Fvclone or hybridoma clone-derived antibodies of the invention.

(iv) Humanized and Human Antibodies

Various methods for humanizing non-human antibodies are known in theart. For example, a humanized antibody has one or more amino acidresidues introduced into it from a source which is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one embodiment of the method,humanized antibodies are prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Human antibodies of the invention can be constructed by combining Fvclone variable domain sequence(s) selected from human-derived phagedisplay libraries with known human constant domain sequence(s) asdescribed above. Alternatively, human monoclonal antibodies of theinvention can be made by the hybridoma method. Human myeloma andmouse-human heteromyeloma cell lines for the production of humanmonoclonal antibodies have been described, for example, by Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).

It is possible to produce transgenic animals (e.g., mice) that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray in such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge. See, e.g., Jakobovits et al,Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immuno., 7:33 (1993);and Duchosal et al. Nature 355:258 (1992).

Gene shuffling can also be used to derive human antibodies fromnon-human, e.g. rodent, antibodies, where the human antibody has similaraffinities and specificities to the starting non-human antibody.According to this method, which is also called “epitope imprinting”,either the heavy or light chain variable region of a non-human antibodyfragment obtained by phage display techniques as described herein isreplaced with a repertoire of human V domain genes, creating apopulation of non-human chain/human chain scFv or Fab chimeras.Selection with antigen results in isolation of a non-human chain/humanchain chimeric scFv or Fab wherein the human chain restores the antigenbinding site destroyed upon removal of the corresponding non-human chainin the primary phage display clone, i.e. the epitope governs (imprints)the choice of the human chain partner. When the process is repeated inorder to replace the remaining non-human chain, a human antibody isobtained (see PCT WO 93/06213 published Apr. 1, 1993). Unliketraditional humanization of non-human antibodies by CDR grafting, thistechnique provides completely human antibodies, which have no FR or CDRresidues of non-human origin.

(v) Antibody Fragments

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. (2003) Nat. Med.9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is typical to have thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. One interface comprises at least a part of the C_(H) 3 domainof an antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al, J. Immunol, 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

(vii) Single-Domain Antibodies

In some embodiments, an antibody of the invention is a single-domainantibody. A single-domain antibody is a single polypeptide chaincomprising all or a portion of the heavy chain variable domain or all ora portion of the light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domain antibody(Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).In one embodiment, a single-domain antibody consists of all or a portionof the heavy chain variable domain of an antibody.

(viii) Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

(ix) Antibody Derivatives

The antibodies of the invention can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. In certain embodiments, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

(x) Vectors, Host Cells, and Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-antigent antibody, nucleic acidencoding the antibody is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(a) Signal Sequence Component

An antibody of the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (e.g., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native antibody signal sequence, the signalsequence is substituted by a prokaryotic signal sequence selected, forexample, from the group of the alkaline phosphatase, penicillinase, lpp,or heat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO 90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

(b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter).

(c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and -II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(d) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(e) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, α-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to theinvention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NSO andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(h) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(xi) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

D. Selecting Biologically Active Antibodies

Antibodies produced as described above may be subjected to one or more“biological activity” assays to select an antibody with beneficialproperties from a therapeutic perspective. The antibody may be screenedfor its ability to bind the antigen against which it was raised. Forexample, for an anti-VEGF antibody, as shown in the example below, theantigen binding properties of the antibody can be evaluated in an assaythat detects the ability to bind to VEGF.

In another embodiment, the affinity of the antibody may be determined bysaturation binding; ELISA; and/or competition assays (e.g. RIA's), forexample.

Also, the antibody may be subjected to other biological activity assays,e.g., in order to evaluate its effectiveness as a therapeutic. Suchassays are known in the art and depend on the target antigen andintended use for the antibody.

To screen for antibodies which bind to a particular epitope on theantigen of interest (e.g., those which block binding of the anti-VEGFantibody of the example to VEGF), a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

E. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided comprising a container which holds the aqueous pharmaceuticalformulation of the invention and optionally provides instructions forits use. Suitable containers include, for example, bottles, vials andsyringes. The container may be formed from a variety of materials suchas glass or plastic. An exemplary container is a 3-20 cc single useglass vial. Alternatively, for a multidose formulation, the containermay be 3-100 cc glass vial. The container holds the formulation and thelabel on, or associated with, the container may indicate directions foruse. The article of manufacture may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations areincorporated herein by reference.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

Example 1 Stable Anti-VEGF Antibody Liquid Formulations

This examples describe the development and stability testing of stableliquid formulations comprising anti-VEGF antibody at proteinconcentrations in the range from about 20 mg/mL-200 mg/mL in variousliquid formulations comprising histidine, arginine, acetate, or sodiumchloride. One milliliter of each formulation in a 3 cc glass vials wasstored at 40° C. and the stability was assessed at 1, 2, and 4 weeks.The stability of anti-VEGF was monitored by several assays including UV(for concentration and turbidity), size exclusion chromatography (SEC)for size variant analysis, imaged capillary isoelectric focusing (icIEF)for charge variant analysis, CE-SDS for size distribution and bindingassay for activity. After four weeks of stability testing, our resultsindicate that anti-VEGF is stable in 200 mM Arginine Acetate, 150 mMSodium Chloride, 0.04% PS20, pH 5.2.

The stability (e.g., aggregate formation, viscosity, etc.) of anti-VEGFwas investigated in various liquid formulations comprising histidine,sodium chloride, arginine, and acetate. The stability of anti-VEGF wasmonitored by several assays including, size exclusion chromatography(SEC) for aggregate formation analysis. Our results indicate thatanti-VEGF is stable at about pH 5.2 in arginine-containing buffers.

Anti-VEGF was formulated into different buffers by dialysis usingSlide-a-Lyzer® cassettes to achieve the final concentrations listed inTable 1. Each formulation was sterile filtered with 0.22 μm Steriflip®filter units and aseptically filled into autoclaved vials, stoppered,and sealed. Samples were placed at 2-8° C., 25° C., and 40° C. andstability studies were conducted at select temperatures.

TABLE 1 Formulations Formulation A 51 mM sodium phosphate, 159 mMTrehalose, 0.04% PS20, pH 6.2 B 200 mM Arginine Acetate, 0.04% PS20, pH5.2 C 20 mM Sodium Acetate, 240 mM Sucrose, 0.04% PS20, pH 5.2 D 20 mMHistidine Chloride, 200 mM Arginine Chloride, 0.04% PS20, pH 5.2

Methods

pH: A 200 μL volume of each sample was placed in a 1.5 ml Eppendorftubes at an ambient temperature and their pH was measured using ThermoOrion pH meter equipped with a Ross® semi-micro electrode. The pH meterwas calibrated using Thermo Orion buffer standards pH 4.0, 5.0 and 7.0.

Viscosity: Shear viscosity was measured using an Anton Paar PhysicaMCR300 rheometer with a 25 mm cone (CP 25-1) set at a height of 0.049mm. 75 μL of each sample was loaded onto a Peltier plate at 25° C. andmeasured 10 times per 100 s interval at a constant shear rate of 1000l/s.

Size Exclusion Exchange Chromatography (SEC): Size exclusionchromatography was performed to quantitate total aggregate levels (withneat injections) and slow-dissociating aggregate levels (with diluteinjections). Dilute injections were diluted with mobile phase buffer(0.20M potassium phosphate, 0.25M potassium chloride, pH 6.2) to 0.5mg/mL. All samples were incubated at 30° C. for 24 hours prior toanalysis. 10 μL of each neat sample and 100 μL of each dilute samplewere injected onto a TSK G3000SWXL, 7.8×300 mm column (TOSOHAAS, partno. 08541) using an Agilent 1100 HPLC system. The autosampler was keptat 30° C. while the column was kept at ambient temperature. Flow ratewas 0.5 mL/min and total run time per sample was 30 minutes. Data wasanalyzed with sample absorbance at 280 nm using HP Chemstation.

Ion Exchange Chromatography (IEC): Ion exchange chromatography wasperformed to quantitate charged variants in carboxypeptidase B(CpB)-digested samples. Samples were diluted to 1 mg/mL with Solvent A(20 mM N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES) buffer, pH6.5), treated with a 1% w/w addition of 1 mg/mL CpB, and incubated for20 minutes at 37° C. 50 μL of each sample was then injected onto aDionex ProPac WCX-10, 4.6×250 mm column using an Agilent 1100 HPLCsystem. Autosampler temperature was kept at 2-8° C. while the column waskept at 40° C. Flow rate was 0.5 mL/min while using a gradient ofSolvent A and Solvent B (200 mM sodium chloride in Solvent A) over 90minutes, as listed in the test procedure. Data was analyzed with sampleabsorbance at 280 nm using HP Chemstation.

Turbidity Assay: To monitor turbidity the optical density of eachformulation was measured at 350 nm using an Agilent 8453 UV-VISspectrophotmeter. All samples were analyzed without dilution using a 1cm pathlength quartz cuvette.

−20° C. and Freeze Thaw Stability Studies: Formulations A-D areaseptically filled into 316 L stainless steel mini-cans (15 mL/minican).All samples are stored at −20° C. for varying amounts of time (e.g., 24,48, 72, or more hours; 4, 5, 6, 7, or more days; 2, 3, 4, 5, 6, 7, 8, ormore weeks; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, or more months, and continuously sampled asepticallyunder a laminar flow hood. In addition, formulations A-D are filled into6 cc glass vials and stored at −20° C. Each vial is subjected to fivefreeze thaw cycles and analyzed using SEC, IEC and turbidity assays. Thefreeze-thaw cycle entails storage for at least 24 hours at −20° C.followed by storage for at least 24 hours at 5° C.

Results and Discussion

This study investigated the stability (e.g., aggregate formation,viscosity, chemical stability etc.) of different concentrations ofanti-VEGF in an arginine-based formulation. SEC and IEC were used tomonitor stability of anti-VEGF at stressed and accelerated storageconditions. Aggregation and viscosity of the anti-VEGF was measured andset forth in Table 2 below and illustrated in FIGS. 2 and 4.

TABLE 2 physical properties of formulations studied Formulation[Protein] (mg/ml) % Total Aggregate Viscosity (cP) A 25 7 A 30 8 A 10010 A 150 13 B 20 N/D 1.3 B 50 2.6 1.9 B 100 3.8 4.2 B 110 N/D 4.7 B 125N/D 6.1 B 150 N/D 9.5 C 20 N/D 1.6 C 50 3 2.3 C 100 5.3 5.7 C 125 N/D12.4 C 150 N/D 23.5 C 175 N/D 52 D 20 N/D 1.3 D 50 2.1 1.8 D 100 3.4 4.3D 125 N/D 6 D 150 N/D 11.3 D 175 N/D 17.7

Aggregation of all Formulations at 40° C.: The amount of total aggregateand dimer formed in each anti-VEGF formulation after storage for 0, 1,2, and 4 weeks at 40° C. was measured and is set forth in Tables 3, 4,and 5 below and illustrated in FIGS. 1 and 3.

TABLE 3 Total aggregate after storage at 40° C. % Total Aggregate Sample4 weeks A 10.3 B 5 C 7.1 D 10.8

TABLE 4 Total aggregate after storage at 40° C. % Total % Total % Total% Total Aggregate Aggregate Aggregate Aggregate Sample 0 weeks 1 week 2weeks 4 weeks B 3.9 4.7 5.3 6.1 C 5.4 6.8 8.3 8.1 D 3.4 5.2 8.4 13.1

TABLE 5 Dimer after storage at 40° C. % Dimer % Dimer % Dimer % DimerSample 0 weeks 1 week 2 weeks 4 weeks B 3.3 3.9 4.4 5.0 C 4.7 6.0 7.47.1 D 2.8 4.2 6.7 10.8Aggregation of Formulations at 25° C.: The amount of total aggregate anddimer formed in each anti-VEGF formulation (100 mg/ml) after storage for0, 2, 4, and 8 weeks at 25° C. was measured and is set forth in Table 6below.

TABLE 6 Total aggregate after storage at 25° C. % Total % Total % Total% Total Aggregate Aggregate Aggregate Aggregate Formulation 0 weeks 2weeks 4 weeks 8 weeks B 3.9 4.6 4.3 3.4 C 5.4 6.6 6.6 4.8 D 3.4 5.1 6.46.1Aggregation of Formulations at 2-8° C.: The amount of aggregate anddimer formed in each anti-VEGF formulation (100 mg/ml) after storage for0 and 4 weeks at 2-8° C. was measured and is set forth in Table 7 below.

TABLE 7 Total aggregate and dimer after storage at 2-8° C. % Total %Total Aggregate Aggregate % Dimer % Dimer Formulation 0 weeks 4 weeks 0weeks 4 weeks B 3.9 3.8 3.3 3.2 C 5.4 5.7 4.7 5.1 D 3.4 3.5 2.8 2.9Aggregation at various Arginine concentrations at 40° C.: The amount oftotal aggregate formed in each anti-VEGF formulation at various arginineacetate concentrations was measured and is set forth in Table 8 below.

TABLE 8 Total aggregate at various Arginine acetate concentrationsArginine Acetate % Total Concentration (mM) Aggregate 25 5.2 50 4.8 1004.4 200 4.2

Effect of Excipients and Ionic Strength: The effect of differentexcipients on the stability of anti-VEGF was investigated. A list ofexcipients explored includes sodium phosphate, arginine acetate, sodiumacetate, and histidine chloride. Our results showed that formulationscontaining arginine chloride and histidine chloride aggregated fasterthan all other formulations.

The stability of anti-VEGF was evaluated in various buffer conditions.The data obtained from the study showed that anti-VEGF is more stable inarginine acetate buffers between pH 4.0 and pH 6.0. The data obtainedfrom the formulation screening study showed that anti-VEGF is stable andhas reduced aggregate and dimer formation at 100 mg/mL proteinconcentration in 200 mM Arginine Acetate, 0.04% PS20 at pH 5.2.

1. A stable aqueous pharmaceutical formulation, the formulationcomprising a therapeutically effective amount of an antibody in anarginine buffer, pH 4.0 to 6.0.
 2. The formulation of claim 1, whereinthe buffer is an arginine acetate buffer, pH 4.5 to 5.5.
 3. Theformulation of claim 1, wherein the buffer is an arginine acetatebuffer, pH 4.8 to 5.4.
 4. The formulation of claim 1, wherein the bufferis an arginine acetate buffer, pH 5.2.
 5. The formulation of claims 2,3, or 4 wherein the arginine actetate concentration in the buffer isfrom about 25 mM to about 250 mM.
 6. The formulation of claims 2, 3, or4 wherein the arginine actetate concentration in the buffer is fromabout 50 mM to about 250 mM.
 7. The formulation of claims 2, 3, or 4wherein the arginine actetate concentration in the buffer is from about75 mM to about 250 mM.
 8. The formulation of claims 2, 3, or 4 whereinthe arginine actetate concentration in the buffer is from about 100 mMto about 250 mM.
 9. The formulation of claims 2, 3, or 4, wherein thearginine acetate concentration in the buffer is from about 120 mM toabout 240 mM.
 10. The formulation of claims 2, 3, or 4, wherein arginineacetate concentration in the buffer is from about 150 mM to about 225mM.
 11. The formulation of claims 2, 3, or 4, wherein the arginineacetate concentration in the buffer is about 200 mM.
 12. The formulationof claim 1, further comprising a surfactant.
 13. The formulation ofclaim 12, wherein the surfactant is polysorbate.
 14. The formulation ofclaim 13, wherein the polysorbate is polysorbate
 20. 15. The formulationof claim 12, wherein the surfactant concentration is from 0.0001% toabout 1.0%.
 16. The formulation of claim 12, wherein the surfactantconcentration is from about 0.01% to about 0.05%.
 17. The formulation ofclaim 12, wherein the surfactant concentration is 0.04%.
 18. Theformulation of claim 1, wherein the antibody concentration is from about10 mg/ml to about 250 mg/ml.
 19. The formulation of claim 1, wherein theantibody concentration is from about 25 mg/ml to 200 mg/ml.
 20. Theformulation of claim 1, wherein the antibody concentration is from about50 mg/ml to about 150 mg/ml.
 21. The formulation of claim 1, wherein theantibody concentration is from about 75 mg/ml to about 125 mg/ml. 22.The formulation of claim 1, wherein the antibody is not subject to priorlyophilization.
 23. The formulation of claim 1 wherein the antibodybinds VEGF.
 24. The formulation of claim 1, wherein the antibody is amonoclonal antibody.
 25. The formulation of claim 24 wherein themonoclonal antibody is a full length antibody.
 26. The formulation ofclaim 24 wherein the monoclonal antibody is an IgG1 antibody.
 27. Theformulation of claim 24 wherein the monoclonal antibody is a humanizedantibody.
 28. The formulation of claim 24 wherein the monoclonalantibody is an antibody fragment comprising an antigen-binding region.29. The formulation of claim 28 wherein the antibody fragment is a Fabor F(ab′)2 fragment.
 30. The formulation of claim 24 wherein themonoclonal antibody binds VEGF.
 31. The formulation of claim 30 whereinthe antibody is bevacizumab.
 32. The formulation of claim 1 wherein themonoclonal antibody is susceptible to aggregation.
 33. The formulationof claim 2 wherein the buffer is 200 mM arginine acetate pH 5.2, thesurfactant is polysorbate in an amount of about 0.01-0.1% v/v, whereinthe formulation is stable at a temperature of about 40° C. for at least28 days
 34. An article of manufacture comprising a container holding astable aqueous pharmaceutical formulation comprising a therapeuticallyeffective amount of an antibody, an arginine acetate buffer from aboutpH 4.5 to about 6.0, and a surfactant.
 35. The article of manufacture ofclaim 34, wherein the antibody binds VEGF.
 36. The article ofmanufacture of claim 35, wherein the antibody is bevacizumab.
 37. Amethod for stabilizing an antibody in an aqueous pharmaceuticalformulation by combining a therapeutically effective amount of anantibody, an arginine acetate buffer from about pH 4.5 to about 6.0, anda surfactant.
 38. The method of claim 37, wherein the antibody bindsVEGF.
 39. The method of claim 38, wherein the antibody is bevacizumab.40. A stable aqueous pharmaceutical formulation comprising atherapeutically effective amount of an antibody, 200 mM arginine acetatebuffer at pH 5.2, and a surfactant.
 41. The formulation of claim 40wherein the antibody binds VEGF.
 42. The formulation of claim 41 whereinthe antibody is bevacizumab
 43. An article of manufacture comprising acontainer holding the formulation of any one of claims 40-42.
 44. Theformulation of claim 1 which is sterile.
 45. The formulation of claim 1which is stable upon storage at about 40° C. for at least 28 days. 46.The formulation of claim 1 which is aqueous and is administered to asubject.
 47. The formulation of claim 46 wherein the formulation is forintravenous (IV), subcutaneous (SQ) or intramuscular (IM)administration.
 48. The formulation of claim 46, which is for Wadministration and the antibody concentration is from about 10 mg/ml toabout 250 mg/ml.
 49. The formulation of claim 46, which is for IVadministration and the antibody concentration is from about 50 mg/ml toabout 100 mg/ml.
 50. The formulation of claim 46, which is for SQadministration and the antibody concentration is from about 25 mg/ml toabout 250 mg/ml.
 51. The formulation of claim 46, which is for SQadministration and the antibody concentration is from about 50 mg/ml toabout 100 mg/ml.
 52. A vial with a stopper pierceable by a syringecomprising the formulation of claim 1 inside the vial.
 53. The vial ofclaim 52 which is stored at about 2-8° C.
 54. The vial of claim 52 whichis a 3 cc, 20 cc or 50 cc vial.
 55. A stainless steel tank comprisingthe formulation of claim 1 inside the tank.
 56. The tank of claim 55wherein the formulation is frozen.
 57. A method of treating a disease ordisorder in a subject comprising administering the formulation of claim1 to a subject in an amount effective to treat the disease or disorder.58. The method of claim 57 wherein the antibody binds VEGF.
 59. Themethod of claim 58 wherein the antibody is bevacizumab
 60. Apharmaceutical formulation comprising: (a) a full length IgG1 antibodysusceptible to deamidation or aggregation in an amount from about 10mg/mL to about 250 mg/mL; (b) arginine acetate buffer, pH 4.5 to 6.0;and (c) polysorbate 20 in an amount from about 0.01% to about 0.1%. 61.The formulation of claim 60 wherein the antibody binds VEGF.
 62. Theformulation of claim 61 wherein the antibody is bevacizumab
 63. A methodfor reducing aggregation of a therapeutic monoclonal antibody,comprising formulating the antibody in an arginine acetate buffer, pH4.5 to 6.0.
 64. The method of claim 63 wherein the antibody binds VEGF.65. The method of claim 64 wherein the antibody is bevacizumab.
 66. Apharmaceutical formulation comprising an antibody that binds to VEGF inan arginine acetate buffer at a pH from about 4.5 to about 6.0, and asurfactant.
 67. The formulation of claim 66 wherein the antibody isbevacizumab.
 68. A method of making a pharmaceutical formulationcomprising: (a) preparing the formulation of claim 1; and (b) evaluatingphysical stability, chemical stability, or biological activity of theantibody in the formulation.
 69. The method of claim 68, wherein theantibody binds VEGF.
 70. The method of claim 69, wherein the antibody isbevacizumab